Gender Differences in the Long-Chain Polyunsaturated Fatty Acid Status: Systematic Review of 51 Publications

Long-chain-polyunsaturated fatty acids (LCPUFA) have important functions in cell membranes as indispensable building stones for human development and optimal health. Docosahexaenoic acid (DHA) and arachidonic acid (AA) are considered to be the most important functional LCPUFA. They can be provided directly from the diet or can be synthetized from their essential fatty acid precursors α-linolenic acid (ALA) and linoleic acid (LA). Among other enzymes, Δ⁶- and Δ⁵-desaturases are required for the formation of the longer-chain metabolites of both n–3 and n–6 series. The competitive desaturation of the n–3 and n–6 series of fatty acids by Δ⁶-desaturase is of major significance, because this step is considered to be the rate-limiting step of the pathway. The activity of this enzyme is modulated by hormones and by interactions of substrates and metabolic products [1,2].

The role of diet in influencing the proportion of fatty acids in plasma, blood cells and adipose tissue has been described in detail previously [3]. However, there are only narrative overviews [4], but no systematic review on the role of the most basic non-dietary determinant, gender, in influencing the fatty acid composition of different tissues. In order to investigate whether LCPUFA metabolism can be eventually influenced by sexual hormones, we decided to systematically review the gender-specific differences between women and men in the contribution of LCPUFA to the fatty acid composition of different biological samples.

Plasma phospholipids (PL), plasma cholesteryl esters (CE), plasma triacylglycerols (TG), total plasma lipids, erythrocyte and platelet lipids as well as subcutaneous adipose tissue from the abdomen or buttock are the lipid pools most often reported in the literature for the characterization of fatty acid status. Therefore, we decided to review data on gender differences in the fatty acid composition of these biomarkers.

The aim of this systematic literature review was to compare LCPUFA status on the basis of the fatty acid composition of different biomarkers in healthy males and females.

To be included into the review, a study needed to meet all of the following characteristics: (1) a study carried out in humans; (2) at least 14 participants included; (3) n–3 or n–6 LCPUFA status is reported in both males and females; (4) healthy individuals with normal weight were included, or population-based surveys were carried out in that the majority of participants were considered healthy; (5) omnivorous participants were included; (6) there was no dietary intervention (especially no lipid-modified diet) or drug therapy before sample collection, and (7) investigators measured at least 12 fatty acids by gas liquid chromatography, so the percentage distribution data contained the principal fatty acids of the fatty acid spectrum and presumably reflected a realistic proportion of fatty acids in the given lipid fraction.

Electronic Searches

Ovid MEDLINE (www.ovid.com), Scopus (www.scopus. elsevier.com) and the Cochrane Library central database (www.thecochranelibrary.org) were searched from inception to February 2011 for studies containing LCPUFA values of both men and women (boys and girls) using text terms with appropriate truncation and relevant indexing terms. The search was in the form [n–3 LCPUFA terms] or [n–6 LCPUFA terms] and [biomarker terms] and [gender terms] and [differ*] and [human studies]. The results obtained by the full Ovid MEDLINE search strategy are shown in online supplementary table 1 (for all online suppl. material, see www.karger.com?doi=10.1159345599). The searches of the two other databases were also based on this strategy. We did not apply any language restriction.

Titles and abstracts found by the electronic and bibliographic searches were screened for inclusion by a single reviewer (S.L.). Due to the large number of papers which were present in more than one database, duplications were filtered out to compile the final list of titles and abstracts to be screened. Thereafter, it was checked which of the titles and abstracts met the predetermined eligibility criteria. If articles seemed to meet the inclusion criteria, or the title and the abstract left room for doubt, the full text of the article was evaluated by two independent reviewers (S.L. and K.F.). If the two reviewers disagreed about the eligibility, the study was discussed in detail to reach a consensus decision.

Data for each study included were extracted by a single reviewer (S.L.) into a Microsoft Office Excel 2007 database file. To provide a standardized format, units of measurement were recalculated to percentage contribution of LCPUFA to total fatty acid composition of the relevant tissue (% weight/weight) from the original data in the publication. If it was not possible to convert data, we tried to contact the authors. If the authors could not be contacted (in most of the cases because of the long time elapsed since the publication of the papers), or the data were not available in the original form any more, those studies were excluded. The original forms in which fatty acid data in the included studies were expressed are shown in table 1. In some studies, the investigators measured the fatty acid composition in plasma, in other studies in serum. Because we feel that there is no major difference in the percent fatty acid composition of plasma and serum, for the uniformity of discussion, we used the term ‘plasma’ throughout the paper.

Statistical analyses were performed using the Review Manager 5.1 software (Cochrane Collaboration, Oxford, UK). Mean differences (MD) were used for the analysis of continuous data. The confidence interval (CI) was established at 95%. Values of p < 0.05 were considered to indicate statistical significance. Statistical heterogeneity was assessed using I² statistics (I² of 50% or more indicating presence of heterogeneity).

Study Inclusion

The flow diagram of the literature search for this review is shown in figure 1. Altogether 7,925 titles and abstracts were identified via the electronic search or were found in the reference lists of the review articles. Three hundred and fifty-six of them appeared to be potentially relevant, so we attempted to collect them as full text articles. Thirteen full text articles (3.6%) could not be collected even after repeated attempts by the help of professional librarians, but 343 full text articles were available for detailed assessment for inclusion. Reasons for excluding studies are presented in figure 1. In articles where the investigators analyzed the fatty acid composition of more than one biomarker (for example, plasma and erythrocyte membrane lipids), data were analyzed as originating from 2 separate studies. In some studies, the investigators divided the study population into subgroups in parallel (for example, according to age or geographical location). These subgroups were also included into our review as separate studies. Finally, 91 comparisons reported in 51 publications fulfilled the inclusion criteria.

Among the studies reviewed, there were some methodological differences in the analytical methods used for the determination of fatty acid composition of the different biomarkers. The blood samples collected were stored in a deep-frozen state, but the storage temperature was different (–20, –30, –40, –70 or –80°C). Lipid extraction was carried out by chloroform and methanol in most, but not all the studies. The separation of different lipid fractions was performed by thin layer chromatography. Fatty acid analysis was carried out by gas liquid chromatography in all studies. In some studies, a packed column was used instead of capillary columns (capillary column: 34 papers; packed column: 10 papers, and column type not reported: 7 papers).

We found 11 publications analyzing plasma PL, whereas 8 analyzed plasma CE, 5 plasma TG, 18 total plasma lipids, 9 total erythrocyte membrane lipids, 1 platelets and 7 adipose tissue fatty acid composition. A description of biomarkers identified in 3 or more studies, including also the number of studies, subgroups and participants from both genders, and the results of the primary analysis (MD, I²) are presented in table 2. We discuss in detail only these biomarkers. Descriptive data of biomarkers detected in less than 3 studies are presented in table 1.

In this study, we focused on the following 8 PUFAs: LA, γ-linolenic acid (GLA), dihomo-GLA (DHGLA) and AA from the n–6 series and ALA, eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA) and DHA from the n–3 series. In table 1, we list the fatty acids from these 8 if the authors included the values into the publication.

Primary analysis showed significantly higher contribution of the n–6 essential fatty acid, LA, and the n–6 long-chain metabolite, AA, to plasma total lipids of women compared to men (fig. 2; table 2). As to n–3 fatty acids, the values of the principal LCPUFA, DHA, were significantly higher (fig. 3), while the values of its precursor, DPA, were significantly lower in women compared to men (table 2). However, with the exception of LA and DHA, considerable heterogeneity was seen among the results of the individual studies.

To filter out the potential effect of age, we tried to classify the individual studies into three age categories: 0–12 (a), 13–50 (b) and ≥51 years (c). In the case of plasma total lipids, classification according to age categories yielded sufficient numbers of studies to evaluate the effect of gender in the two adult groups only. Among the long-chain metabolites, in the group aged 13–50 years only the values of DHA (MD: –0.16; 95% CI: –0.26, –0.06; 2,418 participants; I² = 45%) were significantly higher in women, while the values of DPA were significantly lower in women compared to men (MD: 0.07; 95% CI: 0.03, 0.01; 1,130 participants; I² = 67%). In the group aged ≥51 years (containing postmenopausal women), the values of the n–6 LCPUFA, DHGLA (MD: –0.14; 95% CI: –0.24, –0.04; 137 participants; I² = 19%) and AA (MD: –0.25; 95% CI: –0.43, –0.08; 892 participants; I² = 0%) were significantly higher in women compared to men, whereas DHA values did not differ between the two genders.

To consider the potential effect of diet, we classified the studies based on the results of Meyer [56] according to fish eating habits, assigning the Inuit of Nunavik and the Japanese as high n–3 LCPUFA intake group and all other people as low n–3 LCPUFA intake group. In the high fish (n–3 LCPUFA) intake group, values of AA were significantly higher in women compared to men (MD: –0.23; 95% CI: –0.44, –0.02; 2,291 participants; I² = 68%), while there was no significant difference in the DHA levels between the two groups (MD: –0.00; 95% CI: –0.12, 0.12; 2,291 participants; I² = 17%). In the low fish intake group, both AA (MD: –0.24; 95% CI: –0.41, –0.07; 1,383 participants; I² = 8%) and DHA (MD: –0.24; 95% CI: –0.31, –0.16; 1,598 participants; I² = 11%) were significantly higher in women.

Plasma PL compositional data were reported for the largest number of men (n = 4,097) and women (n = 4,444). There was 1 cross-sectional study among the articles [42] included that contained 16 subgroups stratified according to geographic areas; we were able to include 14 subgroups (in one area only women were recruited, whereas another subgroup had to be excluded, because there were also vegans among the participants). Primary analysis revealed significantly higher contribution of both AA (MD: –0.42; 95% CI: –0.65, –0.18; 7,769 participants; I² = 81%) and DHA (MD: –0.37; 95% CI: –0.51, –0.24; 8,541 participants; I² = 79%) to the fatty acid composition of plasma PL in women (fig. 4, 5), while in LA and ALA there was no gender difference. There were not enough studies to carry out subgroup analysis either by age or fish eating habits.

Plasma CE fatty acids were reported in 8 publications. There was no gender difference in AA and DHA values (table 2), but GLA and DHGLA were found significantly higher in men as compared to women.

Five publications reported fatty acid composition of plasma TG in both men and women. The primary analysis showed no difference between both sexes in any of the fatty acids discussed (table 2).

There were 9 publications reporting fatty acid composition of the erythrocyte membrane total lipids in both sexes. The primary analysis showed significant differences only in DHA values, which were higher in women than in men (table 2). Because most of these studies were carried out in participants older than 50 years, in spite of the apparent abundance of data (12 studies with 1,224 participants), there was no possibility to carry out subgroup analysis either by age or fish eating habits.

There was 1 paper reporting erythrocyte phosphatidylcholine (PC) and phosphatidylethanolamine (PEA) fatty acid composition in 5 subgroups [24]. In erythrocyte PC the values of EPA were significantly higher in women than in men, whereas in erythrocyte PEA there was no difference between the two sexes in any of the fatty acids discussed (table 2).

In adipose tissue the values of LA and DHGLA were significantly higher in women than in men, while the values of GLA and AA, and from the n–3 series the values from EPA, were significantly higher in men than in women.

Endogenous synthesis of AA and DHA from their essential fatty acid precursors, LA and ALA, requires the contribution of elongases and desaturases. Since the elongation steps are rapid, whereas the desaturation steps are slower, the latter are considered as the rate-limiting steps. In this systematic review, we found higher contributions of LCPUFA (AA and DHA) to both plasma total lipids and plasma PL in women than in men. We also found significantly higher values of DHA, but not of AA, in women than in men in erythrocyte lipids. It may be assumed with good reason that the higher AA and DHA values found in plasma PL of women may be due to the higher activity of desaturases, especially that of Δ⁶-desaturase.

The effects of sex hormones on essential fatty acid metabolism in humans have already been reviewed in detail by Childs et al. [57]. In short, human studies demonstrated that males and females differ in their ability to synthesize n–3 LCPUFA from ALA, leading to the higher circulating concentrations of DHA in women than in men [57]. In the same review, a significant relationship between plasma and tissue fatty acid composition and circulating sex hormone concentrations was seen, suggesting that estrogen stimulates, whereas testosterone inhibits, the conversion of essential fatty acids into their longer-chain metabolites. This is consistent with findings from both animal studies [58] and human stable-isotope studies, which also showed that women have a higher capacity than men to synthesize DHA from ALA [1,59].

We also tried to evaluate the influence of age on the gender differences in fatty acid composition of biological samples. Since previously several authors suggested that gender differences seen in fatty acid compositional data may be due to the higher estrogen levels in women, we decided to make subgroups according to the presumably changing estrogen levels of women in the course of their life cycle: 0–12 (a), 13–50 (b) and 50–75 years (c). In serum total lipids, the higher levels of LCPUFA in women than in men were also seen in the oldest age group, where mostly postmenopausal women were present. This finding on its own may indicate that not only the higher estrogen levels are responsible for the sex differences in LCPUFA values observed, e.g. in addition to the physiological and hormonal changes caused by aging, different age groups can have very different dietary habits, which may also influence the composition of serum lipids. Moreover, many postmenopausal women receive estrogen supplementation therapy, which in fact may further modify the picture. The data obtained in the present study indicate that gender differences in fatty acid status may be relevant also in the elderly.

Diet is considered as the major factor influencing fatty acid composition in tissues. Plasma PL fatty acid composition represents the dietary intake of fatty acids over periods of weeks or months before sample collection [60,61], while the rate of changes in red blood cells is slower than that seen in plasma lipids [62,63]. The adipose tissue fatty acid pattern represents the diet ingested in the previous 1–2.5 years [64,65]. In the vast majority of the studies included into our review, the composition of diets was not investigated, so it is not possible to tell to what extent dietary fatty acids influence our results. However, there were enough studies to carry out a subgroup analysis in the total plasma lipid fraction by n–3 LCPUFA intake characteristic for the investigated populations. This analysis resulted in an appreciable decrease in the degree of heterogeneity, which confirms the important role of diet in determining LCPUFA status. In the high DHA intake group (including Japanese and Inuit subjects), no significant difference in DHA values was seen between men and women . This observation suggests that gender is a significant potential confounding variable mainly in populations with low dietary n–3 intake.

We think that our systematic review has some strongpoints. Firstly, we were able to identify a considerable number of studies investigating a relatively great number of women and men. Secondly, the studies included in the present review originated from a wide diversity of geographic locations; consequently, the results obtained may be applied without serious geographical restriction. Thirdly, the studies analyzed were carried out during more than 3 decades, so the phenomena observed do not seem to be changing over time. However, there are also some weaknesses of our study. Firstly, studies included were mostly observational studies and not randomized controlled trials, so the quality control potential of the studies included into the review was limited. Secondly, in case of plasma PL, 14 data sets (subgroups) originated from the same cross-sectional study, so these data influenced the statistical results notably. However, these subgroups represented 14 different geographic areas, and so there are good arguments for handling them as different studies. Thirdly, the analytical methods used by the different research groups were not standardized. However, methods of fatty acid analysis are not yet as rigorously standardized as many other laboratory methods, so any systematic review of fatty acid data faces the same difficulty. It is also important to add that there are several factors which influence fatty acid metabolism and differ between genders (e.g. dietary fatty acid intake, alcohol ingestion, relative body fatness or level of physical activity). These factors may all contribute to the gender differences observed in fatty acid composition of different biological samples; however, the main objective of the present review was not to investigate the potential contribution of these factors, but to draw attention to the fact that gender-related differences exist.

In supplementation studies reporting fatty acid composition in serum PL, serum total lipids or erythrocyte membrane lipids, gender distribution should be regarded as significant potential confounding variable.

This study was financially supported by the Hungarian National Research Fund (OTKA K77899) and the European Communities 7th Framework Program (NUTRIMENTHE FP7 – 212652). This paper does not necessarily reflect the views of the Commission of the European Communities and in no way anticipates the future policy in this area.

The authors confirm that there are no financial or other relationships which might lead to a conflict of interest in the publication of this work.